yrimidinyl Prodrugs for Malaria Significance: Malaria parasites infect about a billion individuals and cause up to 2 million deaths per year. The present application is to support translational research leading to the development of a novel and more effective drug strategy to help reduce or eliminate morbidity and mortality from malaria. Promising hit: Over the last two decades, the PI has systematically considered multiple ways to attack malarial thymidylate synthase with nanomolar-level enzyme inhibitors active in whole cell assays. Based on practical issues related to potency, safety, cost of goods, low propensity for resistance, and lack of cross-resistance to other antimalarials, 6-carboxy-2,4-dioxo-5- fluoropyrimidine (5-fluoroorotate or PYP1) stands out as the best hit. It has an EC50 of 5 nM against all malaria parasites tested and is 1,000 less toxic to mammalian cells. Resistance does not occur at relevant doses. Mouse models for malaria and quantitative in vitro studies of safety versus potency suggest that PYP1, as is, has comparable selectivity to pyrimethamine. It can be even better with some improved pharmacokinetics and controlled drug release. Paths to a drug candidate: The PI proposes a hit-to-lead (HTL) plan to deliver a candidate for advancement to preclinical studies. The project will have the following specific aims: (i) Based on recent advances in prodrug chemistry, a focused combinatorial library will be generated with the aim of selective enzyme-based release of active molecule in the parasite but not host cells. (ii) Based on endoperoxide chemistry, trioxanes and tetraoxane derivatives of PYP1 will be made to selectively release the active carboxylic acids in the parasite (iii) Cell-based screens and toxicity panels will be implemented and used to identify appropriate PYP1 precursors and to prioritize them (iv) ADME and PK studies in rats and mice will be used to optimize advancing molecules. (v) The team will seek synergy partners, test for avoidance of resistance and cross-resistance, perform a 5-day rat toxicology and a dog PK study. Concluding goal: Based on 15-years of NIH basic research and the present translational effort, we will nominate a drug candidate and a backup compound. In consultation with NIH program staff, federal or non-profit organizations will be approached to carry the nominated compound through advanced pre-clinical toxicology and formulation process in preparation for first-in-human (FIH) use.